Site-directed mutagenesis involves the use of primer sequences that anneal to a template DNA sequence, targeting a desired area for mutation. Polymerase chain reaction (PCR) methods allow for amplification of genomic targets in sufficient numbers for transformation.
This protocol uses MilliporeSigma's KOD Xtreme™ Hot Start DNA Polymerase, which allows for high fidelity PCR using a simple three-step thermocycle. It is important to complete the entirety of this protocol in one go, rather than delay performing the PCR, digestion, or transformation. From beginning to end, site-directed mutagenesis using this protocol can be completed in approximately 5 hours, with results from the transformation immediately available the next day.
After PCR, DpnI digestion cleaves any plasmids with methylated sites. This selects for the mutated plasmids (which are unmethylated) by destroying any remaining template DNA that were not mutated in the PCR. The DpnI from NEB is designated as a Time-Saver™ restriction enzyme that can sufficiently digest the PCR product in 15 minutes. However, it is safe to digest for longer periods of time and an incubation period of 1 hour is recommended for this protocol.
In mutagenesis, the final mutated plasmid after PCR and DpnI digestion is generally low in concentration. Therefore, high-efficiency transformation methods must be used with ultra-competent DH5α cells to achieve colonies containing the desired plasmid. The entirety of the PCR product should be used when mixing with the competent cells, and SOC medium is highly recommended for all components of the transformation (outgrowth and plate) over LB medium. Additionally, although high-throughput transformation protocols allow for some steps to be skipped, it is recommended that both heat shock and outgrowth in SOC medium are performed for mutagenesis.
Component | Volume | ||
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KOD Xtreme™ Buffer, 2X | 25 μL | ||
Autoclaved Milli-Q® water | 10 μL | ||
dNTPs, 2 mM | 10 μL | ||
Template DNA, 25 ng/μL | 2 μL | ||
Forward primer | 1 μL | ||
Reverse primer | 1 μL | ||
* KOD Xtreme™ Hot Start DNA Polymerase, 1.0 U/μL | 1 μL |
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Total Volume | 50 μL |
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* NOTE: Always add the KOD Xtreme™ Hot Start DNA Polymerase as the last component, immediately before use in thermocycler. Keep the polymerase in the freezer until needed. |
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* NOTE: Adjust time according to the length of the template DNA. |
If there are issues with mutagenesis, try the following troubleshooting tips: